Single cell, whole genome analysis of the aging human cardiomyocyte

衰老人类心肌细胞的单细胞、全基因组分析

• 批准号: 10326403
• 负责人: Sangita Choudhury
• 金额: $ 87.2万
• 依托单位: BOSTON CHILDREN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-01-15 至 2025-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10326403
• 关键词: Adopted; Adult; Age; Aging; Algorithms; Alleles; Autopsy; Brain; Cardiac; Cardiac Myocytes; Cardiomyopathies; Cardiovascular Diseases; Cardiovascular Pathology; Cell Nucleus; Cell Respiration; Cells; Copy Number Polymorphism; DNA; DNA Damage; DNA Sequence Alteration; DNA sequencing; Data; Development; Disease; Disease Progression; Dropout; Elderly; Environmental Risk Factor; Enzyme-Linked Immunosorbent Assay; Freezing; Genetic; Genome; Genomic DNA; Genomics; Goals; Heart; Heart Diseases; Heart failure; Human; In Vitro; Individual; Inherited; Interphase Cell; Knowledge; Lead; Link; Longitudinal Studies; Malignant Neoplasms; Measures; Methods; Modeling; Mutagens; Mutation; Myocardial dysfunction; Myocardium; Neurons; Newborn Infant; Organ; Oxidative Stress; Pathologic; Patients; Pattern; Phenotype; Ploidies; Point Mutation; Premature aging syndrome; Process; Radiation; Radiation therapy; Research; Resolution; Risk Factors; Role; Sampling; Series; Single Nucleotide Polymorphism; Somatic Mutation; Techniques; Testing; Time; Troponin T; Variant; age related; aged; base; cancer radiation therapy; cardiovascular risk factor; cell type; disorder risk; epidemiology study; genome analysis; genome sequencing; genome-wide; genotoxicity; human old age (65+); innovation; insight; novel; prevent; radiation during childhood; radiation effect; whole genome

Project Summary/Abstract Recent evidence suggests that environmental factors causing somatic mutations during the lifetime have a more crucial role, not only in cancer but also in other common diseases, including heart failure. Recent studies have also shown that somatic single nucleotide variants (sSNV) accumulate even in nondividing cells, such as neurons in the human cortex, resulting in thousands of sSNV per neuronal genome by old age. However, genomic DNA changes in aging cardiomyocytes (CM) remain poorly understood. The accumulation of somatic DNA mutations over time has recently been demonstrated to be a hallmark of aging in many human cell types. The current study aims to determine the landscape and role of somatic mutations in aging and cardiac disease by adopting a new technique that allow deep whole-genome sequencing of DNA isolated from single CM taken from the frozen postmortem heart. The first Aim of this study is to evaluate the somatic mutational burden (sSNVs) in aging CM genome. We will also compare CM mutational burden with postmitotic cells from another organ (neurons) to define differences in accumulation rate during aging. In the second Aim, we will ask what are the mutational signature and the mechanisms of mutation formation in the aging human heart and if the heart mutational signature is different than the brain mutational signature. Further to recapitulate the mutational signature and related phenotype in the heart we will directly induce oxidative stress in an in vitro culture model of primary CMs. The final Aim will focus on evaluating the genotoxic effect of radiation in CMs after childhood radiation therapy and the role of radiation in premature aging. The proposed research is significant for the comprehensive, results-based development of strategies for understanding natural aging and disease progression in the human heart. Together with the planned characterization of mutational signatures, the anticipated results may provide knowledge to develop new strategies for preventing the heart disease associated with aging. The proposed study is only possible because of a series of innovations that are, at this time, uniquely available to our research team, 1) a novel method to isolate single CM nuclei from frozen myocardium based on CM ploidy and nuclei cardiac troponin T expression. 2) A major breakthrough by developing “LiRA” and “PhaseDel” algorithm to call sSNV and sSV confidently from tetraploid cells that considers cell-specific depth distributions of DNA sequencing and allele-dropout rates in scWGS data. For the first time, our study will reveal the landscape of somatic mutations, genomic changes during aging and after radiation therapy in human heart muscle cells in a single-cell resolution. In the long term, this study will provide insights that might allow blocking some of the mutational processes ameliorating age-related myocardial dysfunction.

项目概要/摘要 最近的证据表明，在一生中引起体细胞突变的环境因素对 不仅在癌症中，而且在包括心力衰竭在内的其他常见疾病中也发挥着更重要的作用。 还表明，体细胞单核苷酸变异（sSNV）甚至在非分裂细胞中也会积累，例如 人类皮质中的神经元，在老年时每个神经元基因组中产生数千个 sSNV。 衰老心肌细胞 (CM) 的基因组 DNA 变化仍知之甚少。 随着时间的推移，DNA 突变最近被证明是许多人类细胞类型衰老的标志。 目前的研究旨在确定体细胞突变在衰老和心脏病中的情况和作用 通过采用新技术，可以对从单个 CM 中分离出的 DNA 进行深度全基因组测序 这项研究的第一个目的是评估体细胞突变负担。 我们还将比较 CM 突变负荷与来自另一个细胞的有丝分裂后细胞。 器官（神经元）来定义衰老过程中积累率的差异在第二个目标中，我们会问什么。 是衰老人类心脏中的突变特征和突变形成机制，如果 心脏突变特征与大脑突变特征不同，进一步概括一下突变特征。 心脏中的特征和相关表型我们将在体外培养模型中直接诱导氧化应激 最终目标将集中于评估儿童期后辐射对 CM 的遗传毒性影响。 放射治疗和放射在过早衰老中的作用所提出的研究对于放射治疗具有重要意义。 全面、基于结果的战略的制定，以了解自然衰老和疾病 随着突变特征的计划表征的进展， 预期结果可能为制定预防心脏病的新策略提供知识 所提出的研究之所以成为可能，是因为目前存在一系列创新。 时间，我们的研究团队独有，1）一种从冷冻中分离单个 CM 核的新方法 基于 CM 倍体和细胞核心肌肌钙蛋白 T 表达的心肌 2) 的重大突破。 开发“LiRA”和“PhaseDel”算法，可以从四倍体细胞中自信地调用 sSNV 和 sSV 考虑 scWGS 数据中 DNA 测序的细胞特异性深度分布和等位基因丢失率。 我们的研究将首次揭示衰老过程中和衰老后体细胞突变、基因组变化的情况 从长远来看，这项研究将在单细胞分辨率下对人类心肌细胞进行放射治疗。 提供可能阻止某些改善与年龄相关的突变过程的见解 心肌功能障碍。